Objectives: Studies concerning side effects of chlorhexidine as related to the presence of plaque are scarce. The purpose of this study was to compare the side effects of 0.12% chlorhexidine gluconate (CHX) on previously plaque-free (control group) and plaque-covered surfaces (test group). Methods: This study had a single-blind, randomized, split-mouth, 21 days-experimental gingivitis design, including 20 individuals who abandoned all mechanical plaque control methods during 25 days. After 4 days of plaque accumulation, the individuals had 2 randomized quadrants cleaned, remaining 2 quadrants with plaque-covered dental surfaces. On the fourth day, the individuals started with 0.12% CHX rinsing lasting for 21 days. Stain index intensity and extent as well as calculus formation were evaluated during the experimental period. Results: Intergroup comparisons showed statistically higher (p<0.05) stain intensity and extent index as well as calculus formation over the study in test surfaces as compared to control surfaces. Thus, 26.19% of test surfaces presented calculus, whereas calculus was observed in 4.52% in control surfaces. Conclusions: The presence of plaque increased 0.12% CHX side effects. These results strengthen the necessity of biofilm disruption prior to the start of CHX mouthrinses in order to reduce side effects.